A Single Molecule View of the Rad51-ssDNA Interaction

Abstract

Homologous recombination (HR) represents an essential DNA repair mechanism in living cells. The central molecular complex of HR is the nucleoprotein filament, a DNA-protein complex in which recombinase protein Rad51 is bounded onto single-stranded DNA (ssDNA) in a helical form. Earlier studies have shown that efficient filament formation is critical for correct DNA repair, therefore a detailed characterization of the interaction between Rad51 and ssDNA is essential to understanding homologous recombination.We use a combination of single-molecule fluorescence microscopy, optical tweezers and microfluidics to study the interaction of Rad51 with ssDNA. With this approach, we are able to directly visualize Rad51 filament assembly and disassembly on ssDNA at the single-molecule level.View Large Image | View Hi-Res Image | Download PowerPoint SlideWe have quantitatively assessed each individual step of the Rad51-ssDNA interaction (i.e. nucleation, filament extension and disassembly). Moreover, we investigate the mechanical coupling between the ssDNA template and the reaction kinetics of filament by varying the tension on the DNA molecule. Hence, we have obtained new insight into the reaction pathway of this essential biological system.